Webber S, Whiteley J M
Arch Biochem Biophys. 1985 Feb 1;236(2):681-90. doi: 10.1016/0003-9861(85)90674-5.
The various interactions of rat liver dihydrofolate reductase with two unconjugated 7,8-dihydropteridines, 7,8-dihydrobiopterin and 6-methyl-7,8-dihydropteridine, have been compared with those of 7,8-dihydrofolate and folate. Of particular interest was the reactivity demonstrated by 7,8-dihydrobiopterin because of the potential physiological significance of this reaction both in the regeneration of tetrahydrobiopterin, a cofactor for various biological hydroxylations, and as a step in the biosynthesis of this compound from GTP. Kinetic experiments gave Km values of 0.17, 6.42, and 10.2 microM for 7,8-dihydrofolate, 7,8-dihydrobiopterin, and 6-methyl-7,8-dihydropteridine, respectively, with Vmax = 6.22, 2.39, and 1.54 mumol min-1 mg-1. With folate the enzyme showed high affinity (Km = 0.88 microM) but low Vmax (0.20 mumol min-1 mg-1). The natural cofactor was NADPH and a Km of approximately 0.7 microM was measured with each substrate. The enzyme was activated by both p-hydroxymercuribenzoate and urea when assayed with 7,8-dihydrofolate but was inhibited when 7,8-dihydrobiopterin was the substrate. The pH optimum for dihydrofolate reduction was 4 with enhancement at pH greater than or equal to 5.5 in the presence of 1 M NaCl. Peak activity with 7,8-dihydrobiopterin occurred at pH 4.8; this was shifted to pH 5.3 but was not enhanced by 1 M NaCl. Inhibition with methotrexate was similar whether the enzyme was assayed with either the conjugated or unconjugated 7,8-dihydro derivatives. The rat liver enzyme, highly unstable after purification, was stabilized in the presence of the nonionic detergent, Tween-20 (0.1%); however, the comparative properties toward the conjugated and unconjugated substrates were not altered by this treatment.
已将大鼠肝脏二氢叶酸还原酶与两种未结合的7,8 - 二氢蝶啶（7,8 - 二氢生物蝶呤和6 - 甲基 - 7,8 - 二氢蝶啶）的各种相互作用与7,8 - 二氢叶酸和叶酸的相互作用进行了比较。特别令人感兴趣的是7,8 - 二氢生物蝶呤所表现出的反应活性，因为该反应在四氢生物蝶呤（各种生物羟基化反应的一种辅助因子）的再生过程中以及作为从GTP生物合成该化合物的一个步骤中都具有潜在的生理意义。动力学实验得出，7,8 - 二氢叶酸、7,8 - 二氢生物蝶呤和6 - 甲基 - 7,8 - 二氢蝶啶的Km值分别为0.17、6.42和10.2 microM，Vmax分别为6.22、2.39和1.54 μmol min⁻¹ mg⁻¹。对于叶酸，该酶表现出高亲和力（Km = 0.88 microM）但低Vmax（0.20 μmol min⁻¹ mg⁻¹）。天然辅助因子是NADPH，每种底物测得的Km约为0.7 microM。用7,8 - 二氢叶酸进行测定时，该酶被对羟基汞苯甲酸和尿素激活，但以7,8 - 二氢生物蝶呤为底物时则被抑制。二氢叶酸还原的最适pH为4，在1 M NaCl存在下，pH大于或等于5.5时活性增强。7,8 - 二氢生物蝶呤的峰值活性出现在pH 4.8；此值移至pH 5.3，但1 M NaCl未使其活性增强。无论用结合的还是未结合的7,8 - 二氢衍生物测定该酶，甲氨蝶呤的抑制作用都相似。纯化后高度不稳定的大鼠肝脏酶在非离子去污剂吐温 - 20（0.1%）存在下得以稳定；然而，这种处理并未改变其对结合和未结合底物的比较性质。
